With advancing age the thymus undergoes involution resulting in reduced T lymphopoiesis. This decrease in T cell output from the thymus is thought to have a significant impact on the composition and function of the peripheral T lymphocyte compartment. Previous work from this laboratory has identified an age-sensitive step in the thymocyte differentiation process: the commitment of the earliest pro-thymocyte to the T cell lineage as evidenced by the acquisition of CD25 expression. This step appears to be dependent upon IL-7. Mice which are deficient in either IL-7 or IL-7R-alpha show a specific block at this step of thymopoiesis, resulting in a distribution of thymocyte subsets that greatly resemble those of aged mice. The similarity of subset distribution between these knockout mice and aged mice suggests that an age-related loss of IL-7 signaling is a critical factor in the reduced T cell production of the involuting thymus. The three specific aims of this proposal are designed to test this hypothesis. The first specific aim is to assess how aging impacts the IL-7 signaling pathway by examining the synthesis of IL-7, the quantity and distribution of IL-7 receptor chains, and the levels and activation of Jak 1 and 3 and Stat 5 in early pro-thymocyte subsets. The second specific aim will focus on replacing IL-7 synthesis by means of directed expression in the thymus of aging mice, in order to establish the consequence of such cytokine synthesis on T cell differentiation. A plasmid containing the murine IL-7 coding region under the control of the inducible tetracycline promoter has been constructed and will be used to transfect stromal cells which will subsequently be transplanted into the thymus of middle-aged mice. The third aim is to determine the long-term effect on T lymphopoiesis of completely bypassing the IL-7 signaling step by constitutive Bcl-2 expression in the pro-thymocyte subsets, an approach that has successfully restored T cell differentiation in IL-7-/- and IL-7R-/- mutant mice. It is anticipated that these studies will lead to the development of strategies to enhance T lymphopoiesis and thereby improve immune function in the elderly as well as other immunodeficient adults.